DESCRIPTION: The assimilatory reduction of nitrite to ammonia (sulfite to hydrogen sulfide) is the major source of fixed N and S for the production of protein. The overall reduction process involves the 6-electron reduction to nitrite to ammonia. In spite of the importance of this enzyme, there is little direct evidence for the steps in the reduction mechanism. It will be the aim of this proposed work to: 1) characterize the electron transfer properties of the three oxidation states of the hemoprotein subunit E. coli sulfite reductase in the absence of substrate, and 2) in the presence of substrate elucidate the reduction scheme for the transformation of nitrite to ammonia using direct and mediated electrochemical reduction and oxidation. In addition, electrochemical methods, both direct and mediated, will be used to characterize the pi-cation radical of E. coli sulfite reductase (superoxidized state). Of particular interest to this study is the presence of conformational changes associated with the redox reactions which significantly affect the binding of substrate (conformational gating). Electrochemical methods such as cyclic staircase and square-wave voltammetry are ideally suited to investigate the reactions. In order to reduce the effects of diffusion and increase the analytical signal, the enzyme will be attached to the electrode surface via an antibody/antigen reaction. This approach has been shown to yield high enzyme activity and enable the study of faster homogeneous/heterogeneous reactions. By judicious choice of mediators, ligands and pH, the individual steps in the reduction of nitrite will be determined.